Proteins and peptides play critical roles in metabolism, gene expression, signal transduction, cellular and extracellular structures, which are essential to the survival and/or reproduction of any living organism. Many proteins and peptides are useful in therapeutic and/or diagnostic applications, particularly, when available in pure form. Contaminants often prevent realization of therapeutic and/or diagnostic goals and may endanger the health of a patient.
Protein purification or peptide purification is a significant challenge, especially when large amounts of material are required for therapeutic or diagnostic purposes. Procedures that simply and rapidly provide the protein or peptide of interest in pure form and good yield are highly desirable, regardless of scale.
Frequently, proteins and peptides are produced by recombinant DNA techniques because large amounts of heterologous material can be expressed in bacteria or other host cells. As is well known in the art, recombinant DNA techniques involve transfecting host cells with DNA encoding the protein and growing the cells under conditions favoring expression of the heterologous protein (see, e.g., U.S. Pat. Nos. 4,565,785, 4,673,641, 4,378,921).
Human growth hormone (“hGH”) and antagonists for hGH, (i.e., growth hormone antagonists (“GHA”)) are examples of proteins that can be used in a number of therapeutic applications and that have been produced by recombinant methods. Human growth hormone has been used for the treatment of hypopituitary dwarfism and all conditions resulting from low levels of hGH production, whether such condition is caused by genetic defect, injury or hypophysectomy. Human growth hormone can also improve the recovery rate of burn victims and other hospitalized patients. GHA, on the other hand, has been used to treat acromegaly, a form of gigantism caused by overproduction of hGH (Kopchick et al., U.S. Pat. Nos. 5,681,809, 5,958,879, 5,350,836). Other possible medical uses of GHA are the prevention of retinopathy in diabetes patients and the treatment of cancer patients with tumors overexpressing receptors that bind growth hormone (Clark et al., 1996, WO97/11178).
Reverse phase liquid chromatography (“RP-LC”) and reverse phase high-performance liquid chromatography (“RP-HPLC”) are commonly used to purify molecules such as peptides and proteins, produced by either synthetic or recombinant methods. RP-LC and RP-HPLC methods can efficiently separate closely related impurities and have been used to purify many diverse molecules (Lee et al., “Preparative HPLC,” 8th Biotechnology Symposium, Pt. 1, 593-610 (1988)). Further, RP-LC and RP-HPLC have been successfully used to purify molecules, particularly, proteins on an industrial scale (Olsen et al., 1994, J. Chromatog. A, 675, 101).
Typical eluants for RP-LC and RP-HPLC are ethanol, isopropanol, methanol and acetonitrile. Acetonitrile has been used in the purification of proteins such as insulin (Kroeff et al., 1989, J. Chromatography, 461, 45). However, these solvents are all highly flammable and toxic, which presents a significant safety issue when used on a large scale. Further, acetonitrile frequently has a denaturing effect when used as an eluant for reverse phase chromatography. Disposal of toxic organic solvents, particularly on a process scale, entails significant expenditure.
Thus, there is a need in the art for procedures that selectively separate molecules such as peptides, polypeptides (e.g., hGH and GHA) and organic compounds from impurities by RP-LC and RP-HPLC using inflammable solvents that are less toxic, cheaper and less denaturing than typical solvents used as eluants in RP-LC and RP-HPLC